During the last year, we continued to define the molecular events that regulate hematopoietic stem and progenitor cell (HSPC) quiescence, survival, self-renewal and, myeloid cell lineage commitment and differentiation. We have focused our efforts on transcription factors since they are essential for stem cell growth and differentiation, and are frequently deregulated during the development of leukemias and lymphomas. We previously found that the helix loop helix (HLH) transcription factor, inhibitor of differentiation 1 (Id1), is induced during the early stages of myeloid development, and can instruct hematopoietic stem cells toward a myeloid versus erythroid and lymphoid cell fate suggesting that this gene and other family members Id2 and Id3 may regulate cell specification from HSPC. We have extended these studies to determine if Id1 is required for normal hematopoietic development using Id1-/- mice. Id-/- mice are viable and show no obvious defects. However, these mice have hematopoietic phenotypes including increased hematopoietic stem/progenitor cell cycling and defects in B cell and myeloid cell development. We determined that the hematopoietic phenotype observed in the Id1-/- mice was not intrinsic to the Id1-/- hematopoietic cells, but were due to defects in the microenvironment or niche. The hematopoietic microenvironment is a complex mixture of cells that includes endothelial cells, mesenchymal stem and progenitor cells and their differentiated progeny, adipocytes, osteoblasts, smooth muscle, and chondrocytes. Therefore, we developed and tested a conditional mouse model of Id1, and have initiated studies to specifically delete Id1 in endothelial cells, osteoblasts and other stromal cell populations using a an Id1 conditional mouse model recently developed in our lab, and transgenic mouse strains that express cre recombinase in endothelial, adipocytes and osteoblasts cell lineages. We previously discovered that Id2 is a physiological regulator of B cell and erythroid cell development by negatively regulating functions of E2A and Pu.1 respectively. We have also discovered that Id2 is a direct transcriptional target of Gfi-1, and that Gfi-1 represses Id2 expression in B cell progenitors, which connects Gfi-1 to the B cell transcriptional network via Id genes. Gfi-1 is a transcription factor that is required for lymphoid, myeloid and stem cell development. Over the last year we have discovered that Id2 mediates GFi-1 effects on short term reconstituting cells. In particular, we have rescued the ability of Gfi-1-/- bone marrow cells to rescue irradiated recipient mice by reducing the levels of Id2 in vivo by 50%. Future studies are focused on the mechanism(s) by which Id2 rescues these progenitors, and determine if other Id genes, Id1 and Id3, contribute to the hematopoietic defects observed in Gfi-1 mice. We have found that over expression of Id1, Id2 and Id3 in HSPC results in a myeloproliferative disease (MPD) in mice transplanted with transduced HSPC. In addition, we determined that Id1 and Id2 are over expressed in many AML patient samples suggesting a role for these genes in hematopoietic malignancies. We have recently determined that over expression of Id1 promotes, while knock down of Id1 inhibits the growth of AML cell lines in vitro and in vivo suggesting that Id1 may represent a viable therapeutic target to treat hematopoietic malignancies. Current efforts are focused on determining if knock down of Id1 in AML patient samples can inhibit leukemic cell growth in vitro and in vivo when transplanted into SCID mouse recipients. In an effort to identify novel transcriptional regulators of myeloid cell growth and differentiation, we have compared the global gene expression profile of undifferentiated and differentiating hematopoietic progenitor cells. We have identified a novel zinc finger transcription factor of unknown function, POGZ, which is down regulated during the early stages of myeloid development. We have generated a mouse strain with a targeted deletion of POGZ. POGZ-/- mice do not survive beyond the first few hours of life, and die at birth of unknown causes, suggesting that POGZ is essential for mouse survival. We have discovered that fetal liver hematopoietic cell development is impaired in POGZ-/- mice, which includes a dramatic decrease in cellularity. In addition, fetal thymic development is arrested at a very early stage of development suggesting that POGZ is required for thymic development. We have identified a putative consensus binding site for POGZ, and demonstrated that POGZ can enhance the reporter activity of one gene that contained this site in luciferase reporter assays. Over the last year, the we have collaborated with Dr. Tim Veenstra and Dr. Thorkell Andresson in the Proteomics Core Lab to identify the POGZ interactome using tandem array purification (TAP) and mass spectrometry. POGZ is found in complexes with histone-lysine N-methyltransferases (HMT) G9a and GLP. These interactions were confirmed in co-immunoprecipitation experiments using antibodies that recognize POGZ, G9a, and GLP, and suggest that POGZ is present in a complex that binds both HMT. Furthermore, this interactome suggests that POGZ binds to specific sites on the DNA including promoters, and recruits HMT to di methylate lysine 9 on histone 3 (H3K9me2), a well characterized suppressive epigenetic mark, resulting in repression of gene transcription that contributes to the hematopoietic phenotype observed in the POGZ knock out mouse model. Microarray analysis of WT and KO POGZ fetal liver cells show that 80% of the differentially expressed genes were upregulated, while 20% of the differentially expressed genes were down regulated suggesting that POGZ mainly functions to repress gene expression, although, POGZ may also activate gene expression as indicated above. Future studies are planned to evaluate additional POGZ promoters and to mutate these sites. In addition, studies planned to compare gene expression by micro array analysis of POGZ-/- and POGZ+/+ tissue, with genes that contain the novel consensus POGZ binding site in their promoters, and Chip-seq data sets to identify potential direct targets of POGZ.